Effect And Mechanism Of Trimethylamine Oxide (TMAO) On Vascular Neointimal Hyperplasia After Balloon Injury

Background:Cardiovascular diseases remains the leading cause of mortality in human entered the 21st century.Among them,atherosclerotic heart disease has the most extensive impact on health.The main ways to treating atherosclerotic heart disease is the Percutaneous Transluminal Coronary Intervention(PCI)and aorto-coronary bypass grafting(GAGB).A certain rate of restenosis after surgery is a thorny problem to clinician.Vascular restenosis after PCI and GAGB has the pathological feature of neointimal hyperplasia.Endothelial cell dysfunction and reendothelialization are initiating factors of neointimal hyperplasia.However,the specific mechanism underlying neointimal hyperplasia has not been fully elucidated.Autophagy is considered to be associated with a variety of cardiovascular diseases,including restenosis.Autophagy and Autophagy inhibition are involved in the occurrence and development of diseases.Trimethylamine oxide(TMAO)is a metabolite of dietary choline and other gut microbial metabolites containing trimethylamine nutrients,which is highly correlated with the risk of cardiovascular diseases.TMAO was found to induce endothelial dysfunction and cardiomyocyte hypertrophy,but the relationship between TMAO and Vascular Neointimal Hyperplasia is unclear.Objective:To explore whether TMAO promotes endothelial cell dysfunction and neointimal hyperplasia after balloon injury by affecting autophagic flux and elucidate the underlying mechanism.Methods and Results:1.Effect of TMAO on vascular neointimal hyperplasia after balloon injury.The rat carotid artery balloon injury model was constructed.After the operation,rats were given TMAO,quadruple antibiotics or an equal volume of normal saline intervention,and normal rats were taken as the sham operation group.LC/MS/MS was used to detect the level of TMAO in rat serum.HE staining was performed on vascular tissue sections.PCNA protein immunohistochemical staining was used to further reflect the intimal hyperplasia and CD31 protein immunohistochemical staining was used to reflect the reendothelialization.The expression level of PCNA was detected by western blot.The results showed that increased serum TMAO levels can significantly increase the I/M ratio and the number of PCNA-positive cells in the injured carotid artery tissue,reduce the number of CD31-positive cells,and increase the protein level of PCNA.Then RVSMCs and RAOECs were treated with TMAO(0μM、100μM、300μM、600μM)for 24 or 48h.CCK-8 and EdU was used to detect the proliferation activity of cells.The scratch test and Transwell test was used to detect the migration ability of cells.The detection of ROS level reflects the degree of oxidative stress,the level of NO was measured by Griess and the cell permeability was measured by FITC-Dextron.The protein level of PCNA,Occludin and cyclinDl was measured by western blot.The results showed that TMAO inhibited the cell proliferation and migration function of RAOECs,promoted oxidative stress of RAOECs,destroyed the cell barrier function,and weakened the ability of reendothelialization.However,TMAO had no significant effect on the proliferation,migration and phenotype transition of RVSMCs.2.Mechanism of TMAO on vascular neointimal hyperplasia after balloon injury.The number of autophagosomes in the rat carotid artery balloon injury model after TMAO intervention was detected by electron microscope and the mRFP-GFP-LC3 fluorescent double-label pair was used to measure autophagic flux.The protein level of P62、Beclinl、LC3 and cylinD1 was measured by western blot.Then siRNA was added to downregulate Beclinl in TMAO-treated RAOECs.EdU was used to detect the proliferation activity of cells.The scratch test was used to detect the migration ability of cells.The results showed that TMAO blocked the flux of autophagy in balloon-injured rats and TMAO-treated RAOECs.However,the adverse effects of TMAO on the proliferation and migration of RAOECs were reversed by Beclinl knockdown.It proved that TMAO-impaired autophagic flux promoted RAOECs dysregulation via activating Beclin1.Conclusions:This study proved that TMAO-induced blockage of autophagic flux aggravated neointimal hyperplasia via activate Beclinl signaling pathway after balloon injury.Reducing blood circulation TMAO levels can be a potential treatment strategy for postoperative vascular restenosis.